This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.
Background Information
TLR10 plays a role in recognizing pathogens and activating innate immunity (NCBI). It recognizes "pathogen-associated molecular patterns" (PAMPs) on disease-causing agents and therefore, produces cytokines that are necessary for immunity (NCBI). TLR10 is most highly expressed in lymphoid tissues including the spleen, lymph node, thymus and tonsil (NCBI). TLR10 is most closely related to TLR1 and TLR6 (Chuang & Ulevitch, 2001). Recent research suggests TLR10 plays a role in a range of viral and microbial diseases due to its activation of immune responses following influenza viral infection (Lee et. al., 2014). TLR10 contains 811 amino acid residues and is characterized by a signal peptide followed by multiple leucine-rich repeats (LRRs), a cysteine-rich domain and a cytoplasmic domain homologous to that of the human interleukin-1 receptor (Chuang & Ulevitch, 2001).
Methods/Approach
The human protein sequence for TLR10 (ENSP00000308925) was attained through the Ensembl database. This protein sequence was used as a query in a BLAST against the whale shark protein database with the Georgia Aquarium Galaxy server. Predicted protein hits were chosen, and their sequences were obtained through the Galaxy server as well. These predicted protein sequences were then used as queries in subsequent BLASTs against the human protein database through NCBI.
Orthologs were searched for by using the human protein sequence for TLR10 (ENSP00000308925) as the query sequence for BLASTs against the protein databases of other species.
A phylogenetic tree was created with CLUSTALW using the top predicted protein hits of the BLASTs against the protein databases of other species using the human protein sequence as the query.
Searching for TLR10 in the Whale Shark
The human protein sequence for TLR10 was used as the query in a BLAST against the whale shark protein database through the Galaxy server. The best predicted protein hits are listed in Table 1. Numerous hits are shown in Figure 1.
Figure 1
Figure 1: Results from the BLAST of the human TLR10 protein sequence against the whale shark protein database. The top 4 best hits were chosen.
Table 1
Whale Shark ID
E-value
Alignment Length
Predicted Protein Length
% Identity
g36276.t1
3e-34
317
925
31.23
g21305.t1
9e-24
149
290
34.23
g48010.t1
6e-26
231
244
31.17
g46258.t1
6e-13
126
645
31.75
Table 1: Top predicted protein hits for the whale shark protein database. The human protein sequence for TLR10 was used as a query in a BLAST against the whale shark protein database. E-values, alignment length, and % identity were analyzed in order to select four hits. The specifics for these hits are listed here.
The top four hits from the whale shark genome as shown in Table 1 and Figure 1 were used as queries in BLASTs against the human protein database. The g36276.t1 whale shark sequence returned a toll-like receptor 3 precursor as the best hit with a query coverage of 94%, an e-value of 2e-58, and a percent identity of 28%. The other whale shark sequences returned similar best hits, all of which fell into the TLR family. Therefore, we were somewhat confident that the whale shark contains an ortholog of the human TLR 10 protein.
Protein Domains
All of the best hit results from using the whale shark predicted protein sequence as queries against the human protein database resulted in predicted proteins from the TLR family and the LRR_IR superfamily as seen in Figure 2. These "leucine-rich repeat" units can be found in many proteins. LRR proteins are involved in protein interactions and ligand interactions including "mammalian innate immune response (NCBI)." The TIR (toll/interleukin-1) domain also seemed to be conserved in the whale shark genome. The toll/interleukin-1 receptor domain is involved in intracellular signaling and works to control protein-protein interactions between toll-like receptors and components of the signal-transduction pathway (NCBI)
Figure 2
Figure 2: Putative conserved domains found in the whale shark genome resulting from a BLAST against the human protein database using the predicted TLR10 whale shark sequence as query. The LRR_RI and TIR families and their subsequent domains were prominent among the putative conserved domain results from various BLASTs using the best hits from the whale shark.
Orthologs
The human TLR10 protein sequence was used as the query in subsequent BLASTs against the protein databases of the mouse, zebrafish, clawed frog, yeast, and elephant shark in order to find orthologs. The results are shown in Table 2 where the best hits were chosen for each species. It appears that all of these species except for yeast appear to have TLR10 orthologs.
Table 2
Species
Top Predicted Protein
ID/Accession
Predicted Protein Length
E-value
Query coverage
% Identity
Homo sapiens
Toll-like receptor 10
NP_112218.2
811
N/A
100%
N/A
Mouse
Toll-like receptor 1 precursor
NP_109607.1
795
0.0
96%
50%
Zebrafish
Toll-like receptor 6
NP_001124065.1
795
2e-126
94%
34%
Clawed frog
Toll-like receptor 6-like isoform X1
XP_002938695.1
842
0.0
92%
44%
Yeast
Adenylate cyclase
NP_012529.3
2026
0.001
21%
26%
Elephant shark
Toll-like receptor 1
XP_007887373.1
851
0.0
95%
41%
Table 2: Top predicted protein hits from various species using the human protein sequence as a query. The top hits from each species are listed here.
Phylogeny
A phylogenetic tree was created through ClustalW using the predicted protein sequences of the best hits from different species resulting from the BLASTs using the human TLR10 protein sequence as the query as shown in Figure 3. The whale shark best hits were included as well.
Figure 3
Figure 3: A phylogenetic tree was created using the best hits from different species resulting from a BLAST using the human TLR10 protein sequence as the query. The four best hits from the whale shark protein database were also included as their whale shark IDs. It is interesting to note that 3 of the best hits from the whale sharked group together and are separate from the human, mouse, clawed frog, elephant shark, and zebra fish. Since all of these species have toll-like receptor isoforms, we hypothesized that the whale shark has a form of the TLR protein.
Conclusions
We believe that the whale shark has a toll-like receptor protein. Although it may not specifically have the TLR10 sequence, it does seem to have conserved the TLR protein domain. The result of the cross-blast (using the human TLR10 sequence as query against the whale shark protein database and then using the top predicted protein hits as queries against the human protein database) returned TLR isoforms for all of the whale shark top hits. Therefore, we hypothesize that the whale shark has this protein or an extremely similar one. Therefore, we conclude that the function of the innate immune system in whale sharks may be somewhat similar to that of humans. Further research may look at the specific differences between the TLR10 ortholog in whale sharks and the orthologs in other species. The phylogenetic tree shown in Figure 3 indicates that the whale shark predicted protein sequence is not as similar to the human protein sequence as the mouse, clawed frog, elephant shark, and zebrafish, which cluster together. It is interesting that one of the whale shark best hits has a common ancestor with yeast, which does not seem to have a TLR10 ortholog. Further research may explain why this single whale shark sequence differs from the others whale shark best hits, all of which returned TLR isoforms in BLASTs against the human protein database.
References
Chuang, T., & Ulevitch, R. J. (2001). Identification of hTLR10: A novel human Toll-like receptor preferentially expressed in immune cells. Biochimica Et Biophysica Acta (BBA) - Gene Structure and Expression,1518(1-2), 157-161. doi:10.1016/S0167-4781(00)00289-X
Lee, S. M., Kok, K., Jaume, M., Cheung, T. K., Yip, T., Lai, J. C., . . . Peiris, J. S. (2014). Toll-like receptor 10 is involved in induction of innate immune responses to influenza virus infection. Proceedings of the National Academy of Sciences,111(10), 3793-3798. doi:10.1073/pnas.1324266111
Matsushima, N., & et al. (n.d.). Analyses of Non-Leucine-Rich Repeat (non-LRR) Regions Intervening Between LRRs in Proteins. ScienceDirect. Retrieved from __http://www.sciencedirect.com/science/__article/pii/__S0304416509001895
This Project
This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.
Background Information
TLR10 plays a role in recognizing pathogens and activating innate immunity (NCBI). It recognizes "pathogen-associated molecular patterns" (PAMPs) on disease-causing agents and therefore, produces cytokines that are necessary for immunity (NCBI). TLR10 is most highly expressed in lymphoid tissues including the spleen, lymph node, thymus and tonsil (NCBI). TLR10 is most closely related to TLR1 and TLR6 (Chuang & Ulevitch, 2001). Recent research suggests TLR10 plays a role in a range of viral and microbial diseases due to its activation of immune responses following influenza viral infection (Lee et. al., 2014). TLR10 contains 811 amino acid residues and is characterized by a signal peptide followed by multiple leucine-rich repeats (LRRs), a cysteine-rich domain and a cytoplasmic domain homologous to that of the human interleukin-1 receptor (Chuang & Ulevitch, 2001).
Methods/Approach
The human protein sequence for TLR10 (ENSP00000308925) was attained through the Ensembl database. This protein sequence was used as a query in a BLAST against the whale shark protein database with the Georgia Aquarium Galaxy server. Predicted protein hits were chosen, and their sequences were obtained through the Galaxy server as well. These predicted protein sequences were then used as queries in subsequent BLASTs against the human protein database through NCBI.
Orthologs were searched for by using the human protein sequence for TLR10 (ENSP00000308925) as the query sequence for BLASTs against the protein databases of other species.
A phylogenetic tree was created with CLUSTALW using the top predicted protein hits of the BLASTs against the protein databases of other species using the human protein sequence as the query.
Searching for TLR10 in the Whale Shark
The human protein sequence for TLR10 was used as the query in a BLAST against the whale shark protein database through the Galaxy server. The best predicted protein hits are listed in Table 1. Numerous hits are shown in Figure 1.
Figure 1
Figure 1: Results from the BLAST of the human TLR10 protein sequence against the whale shark protein database. The top 4 best hits were chosen.
Table 1
The top four hits from the whale shark genome as shown in Table 1 and Figure 1 were used as queries in BLASTs against the human protein database. The g36276.t1 whale shark sequence returned a toll-like receptor 3 precursor as the best hit with a query coverage of 94%, an e-value of 2e-58, and a percent identity of 28%. The other whale shark sequences returned similar best hits, all of which fell into the TLR family. Therefore, we were somewhat confident that the whale shark contains an ortholog of the human TLR 10 protein.
Protein Domains
All of the best hit results from using the whale shark predicted protein sequence as queries against the human protein database resulted in predicted proteins from the TLR family and the LRR_IR superfamily as seen in Figure 2. These "leucine-rich repeat" units can be found in many proteins. LRR proteins are involved in protein interactions and ligand interactions including "mammalian innate immune response (NCBI)." The TIR (toll/interleukin-1) domain also seemed to be conserved in the whale shark genome. The toll/interleukin-1 receptor domain is involved in intracellular signaling and works to control protein-protein interactions between toll-like receptors and components of the signal-transduction pathway (NCBI)
Figure 2
Figure 2: Putative conserved domains found in the whale shark genome resulting from a BLAST against the human protein database using the predicted TLR10 whale shark sequence as query. The LRR_RI and TIR families and their subsequent domains were prominent among the putative conserved domain results from various BLASTs using the best hits from the whale shark.
Orthologs
The human TLR10 protein sequence was used as the query in subsequent BLASTs against the protein databases of the mouse, zebrafish, clawed frog, yeast, and elephant shark in order to find orthologs. The results are shown in Table 2 where the best hits were chosen for each species. It appears that all of these species except for yeast appear to have TLR10 orthologs.
Table 2
Phylogeny
A phylogenetic tree was created through ClustalW using the predicted protein sequences of the best hits from different species resulting from the BLASTs using the human TLR10 protein sequence as the query as shown in Figure 3. The whale shark best hits were included as well.
Figure 3
Figure 3: A phylogenetic tree was created using the best hits from different species resulting from a BLAST using the human TLR10 protein sequence as the query. The four best hits from the whale shark protein database were also included as their whale shark IDs. It is interesting to note that 3 of the best hits from the whale sharked group together and are separate from the human, mouse, clawed frog, elephant shark, and zebra fish. Since all of these species have toll-like receptor isoforms, we hypothesized that the whale shark has a form of the TLR protein.
Conclusions
We believe that the whale shark has a toll-like receptor protein. Although it may not specifically have the TLR10 sequence, it does seem to have conserved the TLR protein domain. The result of the cross-blast (using the human TLR10 sequence as query against the whale shark protein database and then using the top predicted protein hits as queries against the human protein database) returned TLR isoforms for all of the whale shark top hits. Therefore, we hypothesize that the whale shark has this protein or an extremely similar one. Therefore, we conclude that the function of the innate immune system in whale sharks may be somewhat similar to that of humans. Further research may look at the specific differences between the TLR10 ortholog in whale sharks and the orthologs in other species. The phylogenetic tree shown in Figure 3 indicates that the whale shark predicted protein sequence is not as similar to the human protein sequence as the mouse, clawed frog, elephant shark, and zebrafish, which cluster together. It is interesting that one of the whale shark best hits has a common ancestor with yeast, which does not seem to have a TLR10 ortholog. Further research may explain why this single whale shark sequence differs from the others whale shark best hits, all of which returned TLR isoforms in BLASTs against the human protein database.
References
Chuang, T., & Ulevitch, R. J. (2001). Identification of hTLR10: A novel human Toll-like receptor preferentially expressed in immune cells. Biochimica Et Biophysica Acta (BBA) - Gene Structure and Expression, 1518(1-2), 157-161. doi:10.1016/S0167-4781(00)00289-X
Genes and mapped phenotypes. (n.d.). Retrieved from http://www.ncbi.nlm.nih.gov/gene/81793Lee, S. M., Kok, K., Jaume, M., Cheung, T. K., Yip, T., Lai, J. C., . . . Peiris, J. S. (2014). Toll-like receptor 10 is involved in induction of innate immune responses to influenza virus infection. Proceedings of the National Academy of Sciences, 111(10), 3793-3798. doi:10.1073/pnas.1324266111
Matsushima, N., & et al. (n.d.). Analyses of Non-Leucine-Rich Repeat (non-LRR) Regions Intervening Between LRRs in Proteins. ScienceDirect. Retrieved from __http://www.sciencedirect.com/science/__article/pii/__S0304416509001895
Toll-Like Receptor 10. (n.d.). Retrieved from GeneCards website: __http://www.genecards.org/cgi-bin/__carddisp.pl?gene=TLR10
Zhu, J., & et al. (n.d.). Characterization of Bovine Toll-like Receptor 8. ScienceDirect. Retrieved
from __http://www.sciencedirect.com/science/article/pii/S0161589008006949__
Websites used for research:
http://www.ensembl.org/index.html
http://www.genome.jp/tools/clustalw/
http://blast.ncbi.nlm.nih.gov/Blast.cgi
http://whaleshark.georgiaaquarium.org/root/index